1. Field of the Invention
The present invention relates to methods and compositions for treating human immunodeficiency virus (HIV) infected individuals by administering to such individuals, compositions which inhibit or prevent replication of the HIV. In particular, the present invention relates to compositions which inhibit or prevent the replicatire and other essential functions of HIV viral protein R (vpr) by competitively binding to the vpr target in human cells, the vpr binding protein (rip-1). More particularly, the present invention relates to compositions which are glucocorticoid receptor (GR) antagonists, which by binding to rip-1, a GR type molecule, prevent vpr from binding to rip-1, and thereby interfere in the essential activities of the complex necessary for HIV replication. In particular, the antagonist prevents translocation of the vpr/rip-1 complex from the cytosol to the nucleus, or signaling of said translocated complex, whereby vpr would otherwise carry on its various activities essential to replication of HIV. The present invention also relates to methods of identifying such compositions which inhibit or prevent replication of HIV.
2. Brief Description of the Prior Art
One approach to treating individuals infected with HIV is to administer to such individuals compounds which directly intervene in and interfere with the machinery by which HIV replicates itself within human cells. With that approach in mind, it is first noted that HIV is a lentivirus whose genome contains only about 9-11 kb of genetic material and less than 10 open reading frames. Thus, each HIV gene is likely to play a vital role in the natural history of the virus in vivo. Further, HIV possesses a collection of small, positive strand open reading frames which encode 1-2 exon genes whose protein products regulate various aspects of the virus' life cycle. Some of these genes are genetic transactivating factors which are necessary for virus replication in all permissive cell types.
The complexity of HIV can thus be attributed to the intricate patterns of regulation of gene expression observed during the vital lifecycle. Since all such regulatory mechanisms are accomplished by the interaction of virally encoded proteins with distinct host cell factors, it is possible to discover compounds which directly interfere with the binding and translocation of those proteins which are essential to replication of HIV.
The progression from HIV infection to AIDS is in large part determined by the effects of HIV on the cells that it infects, including CD4.sup.+ T lymphocytes and macrophages. On the other hand, cell activation, differentiation and proliferation in turn regulate HIV infection and replication in T cells and macrophages. Gallo, R. C. et al. (1984) Science 224:500; Levy, J. A. et al., (1984) Science 225:840; Zack, J. A. et al. (1988) Science 240:1026; Griffin, G. E. et al., (1988) Nature 339:70; Valentin, A. et al. (1991) J. AIDS 4:751; Rich, E. A. et al., (1992) J. Clin. Invest. 89:176; and Schuitemaker, H. et al. (1992) J. Virol. 66:1354. Cell division per se may not be required since HIV and other lentiviruses can proliferate in nonproliferating, terminally differentiated macrophages and growth-arrested T lymphocytes. Rose, R. M. et al. (1986) Am. Rev. Respir. Dis. 143:850; Salahuddin, S. Z. et al. (1986) Blood 68:281; and Li, G. et al. (1993) J. Virol. 67:3969. The ability of lentiviruses, including HIV, to replicate in nonproliferating cells, particularly in macrophages, is believed to be unique among retroviruses and it is significant that several lentiviruses contain a vpr-like gene. Myers, G. et al. (1992) AIDS Res. Hum. Retrovir. 8:373. HIV infection of myeloid cell lines can result in a more differentiated phenotype and increase the expression of factors such as NF-KB which are necessary for HIV replication. Roulston, A. et al. (1992) J. Exp. Med. 175:751; and Chantal Petit, A. J. et al. (1987) J. Clin. Invest. 79:1883.
Since the demonstration in 1987 that the small open reading frame within HIV-1 designated R encodes a 15 KD protein (Wong-Staal, F., et al., (1987) AIDS Res. Hum. Retroviruses 3:33-39), there has been a growing body of literature regarding the function of the virat protein R (vpr). The vpr open reading frame is conserved within all genomes of HIV-1 and HIV-2 and within all pathogenic isolates of simian immunodeficiency virus (SIV) genomes. The evolutionary requirement for economy in design is deemed to require that the presence of vpr in the HIV genome is related to a specific and non dispensable function in the vital life cycle. Furthermore, the vpr protein is the only HIV-1 regulatory gene product which has been shown to be incorporated into virions. This would normally suggest a structural role for vpr, but since vpr deleted viruses are able to produce normal virions, this is deemed to be further evidence of a regulatory role for this molecule.
Further, the presence of vpr in virions has been associated with increased replication kinetics in T lymphocytes, and with the ability of HIV to establish productive infection in monocytes and macrophages. Thus, it has been reported that mutations in the vpr gene result in a decrease in the replication and cytopathogenicity of HIV-1, HIV-2, and SIV in primary CD4.sup.+ T lymphocytes and transformed T cell lines. See, e.g., Ogawa, K., et al., (1989) J. Virol. 63:4110-4114; Shibata, R., et al. (1990a) J. Med. Primatol. 19:217-225; Shibata, R., et al. (1990b) J. Virol. 64:742-747 and Westervelt, P. et al. (1992) J. Virol. 66:3925, although others have reported that mutated vpr gene had no effect on replication (Dedera, D., et al. (1989) Virol. 63:3205-3208).
Importantly, HIV-2 mutated for vpr has been reported unable to infect primary monocyte/macrophages (Hattori, N., et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:8080-8084). Further, viral replication in macrophages may be almost completely inhibited by antisense ribonucleotides targeting the vpr open reading frame. This, together with the induction of rhabdomyosarcoma cellular differentiation, are deemed to dictate a crucial function for vpr in HIV pathogenesis.
The presence of vpr protein in vital particles means an early function for vpr during the infection process, following virus penetration and uncoating. This role is considered to involve vpr interaction with cellular regulatory mechanisms resulting in an increase in cell permissiveness to sustain viral replication processes. See, e.g., Cohen, E. A., et al. 1990a J. Virol. 64:3097-3099; Yu, X. F., et al. (1990) J. Virol. 64:5688-5693.; and, Yuan, X., et al., (1990) AIDS Res. Hum. Retroviruses 6:1265-1271.
Vpr action can involve the upregulation of cellular elements which enhance viral gene expression, or the downmodulation of cellular inhibitory pathways affecting such vital processes. Such cellular disregulation is consistent with the observation that vpr is sufficient for the differentiation and cessation in cellular proliferation of rhabdomyosarcoma and osteosarcoma cell lines. Thus, the vpr gene of HIV-1 has been shown to induce cellular growth inhibition and differentiation in tumor lines of intermediate differentiation in vitro (Levy, D. N. et al. (1993) Cell 72:541). Thus, the ability of a vitally associated protein such as vpr to reinitiate an arrested developmental program is clearly based upon its interaction with other cellular proteins, and since vpr protein originates within viral particles, it is considered that vpr must, accordingly, play a role in establishing productive infection.
In order for vpr to exert its cellular effects, it requires a cellular ligand, which would mediate these functions. There has been no description heretofore of such a ligand, which may also be referred to as a receptor or binding protein, for vpr. Accordingly, there is described herein, as a part of the present invention, the isolation of a 41 KD vpr cytosolic binding protein, rip-1. Vpr and rip-1 coelute in an immunoaffinity system, and can be specifically crosslinked to a 58 KD complex. Using peptide and antibody competition, the site of their interaction has been resolved to amino acids 38 to 60 on the vpr amino acid sequence. Rip-1 has been detected in various cell lines. Rip-1 selectively translocates from the cytosol to the nucleus upon exposure of the cell to vpr either in a soluble form, or through infection with wild type virus, but not in response to PMA, suggesting a coupling in their regulatory functions.
Part of the present invention is the discovery, described in detail further below, of the likelihood that rip-1 is a member of the steroid hormone receptor superfamily, and particularly, that it behaves as a member of the glucocorticoid receptor (GR) family, and more particularly, that it behaves as a GR type II receptor molecule. Thus, it is a key aspect of the present invention to treat individuals infected with HIV, by administering to such individuals compounds which are steroid hormone receptor antagonists, particularly glucocorticoid receptor antagonists, and more particularly GR type II receptor antagonsts. Such receptor antagonists of the present invention will inhibit or prevent the replicatire and other essential functions of vpr by competitively binding to the vpr target in human cells, the vpr binding protein, rip-1.
Perhaps the best known glucocorticoid receptor antagonist is RU-486, or mifepristone. Acting also as a progesterone receptor antagonist, it is a therapeutic abortifacient approved for use in combination with prostaglandins, in Europe and elsewhere. Many other such glucocorticoid receptor antagonists have been described in the literature. While it is possible that RU-486 may have been taken by an individual who was also suffering from an HIV infection at the time, such use would have been purely coincidental, since there has been no suggestion until the present invention that a glucocorticoid receptor antagonist would in any way inhibit or prevent replication of HIV. Moreover, such coincidental use would in all likelihood also have included the concomitant use of prostaglandins, a combination wholly outside the scope of the present invention.
There remains an urgent need to identify methods of treating individuals suffering from HIV infection. There remains a need to identify compounds which prevent or inhibit HIV replication in infected cells and thereby are useful for treating individuals suffering from HIV infection.